We are testing the hypothesis that there are specific proteome changes accompanying stem cell phenotype development. Investigating the differentiation process of stem cells could elucidate the complex events that occur during normal human brain development. Rat bone marrow stromal cells (BMSCs) are multipotent stem cells that are self-renewing with broad potential. When grown in culture, BMSCs can be induced to a neuronal phenotype expressing neuronal marker proteins. Neuronal phenotype expression parallels terminal differentiation, halting cell division. The objective of these studies is to profile proteome changes characteristic of the phenotypic changes. Proteins from the stromal cells will be characterized using two-dimensional gel and mass spectrometric techniques. We are seeking to identify protein biomarkers of neuropsychiatric disorders, such as the obsessive compulsive syndrome that follows streptococcal infections in pediatric patients. A combination of immunoaffinity strategies will be used to separate proteins from patient sera. The mass spectra of peptides from these proteins will be compared to identify those characteristic of patient state and the diesease trait. We have started to use a tandem affinity purification (TAP) strategy to elucidate the required components of the DNA replication complex in yeast. We generated TAP tags targeting the POL12 and TDP1 yeast genes. The POL12 gene TAP complex will confirm the protein interactions associated with a known complex and the TDP1 complex will define proteins tightly associated with (tyr-DNA phosphodiesterase gene). Based upon protein identifications that are made from these affinity purifications, we will target additional yeast genes associated with DNA replication and the TDP1 complex to confirm the associations. Mammalian genes orthologous to those in yeast will then be targeted, and the complexes will be fully characterized both by mass spectrometry. This strategy will be iterative, with each targeted gene providing additional confirmation regarding the synthesome, the role of the tyr-DNA phosphodiesterase and the identification of the component proteins in a functional interaction network.